Heparin like glycosaminoglycans (HLGAGs) are important components of the extracellular matrix that are believed to regulate a wide variety of cellular activities including invasion, migration, proliferation and adhesion. (Dhodapkar, et al. Blood, 91(8):2679-2688, 1998; Woods, et al., Trends in Cell Biology, 8(5):189-192, 1998) HLGAGs accomplish some of these functions by binding to and regulating the biological activities of diverse molecules, including growth factors, morphogens, enzymes, extracellular proteins. HLGAGs are linear polysaccharides characterized by a disaccharide-repeat unit of a uronic acid [α-L-iduronic acid (I) or β-D-glucuronic acid (G)] linked 1,4 to α-D-hexosamine (H). (1) These polymers of 20-100 disaccharide units can be additionally modified through N- and O-sulfation, epimerization at the C5 position of the uronic acid moiety, adding an additional micro-heterogeneity to these information dense molecules. (1.5).
Although the structure and chemistry of HLGAGs are fairly well understood, information on how specific HLGAG sequences modulate different biological processes has proven harder to obtain. The inventors have recently developed a rapid sequencing methodology for polysaccharides using chemical and enzymatic tools to modify or degrade an unknown HLGAG polymer in a sequence-specific manner. (Venkataraman, G., et al., Science, 286, 537-542 (1999), and U.S. patent application Ser. Nos. 09/557,997 and 09/558,137, both filed on Apr. 24, 2000). An important enzymatic tool in this sequencing process is the heparinases, including heparinases I, II and III. The three heparinases are HLGAG degrading enzymes which can be produced by Flavobacterium heparinum. Each of the heparinases has its own unique HLGAG sequence at which it cleaves, making these enzymes valuable tools in obtaining sequence specific information. Heparinase I primarily cleaves HLGAGs at the HNS,6X-I2S2-linkage found primarily in heparin-like regions (Ernst, S., et al., Crit, Rev. Biochem. Mol. Biol., 30, 387-444 (1995)). Desai, U., et al., Biochemistry, 32, 8140-8145 (1993)), and Jandik, K., et al., Glycobiology, 4, 289-296 (1994)). Heparinase III cleaves at the HNAC-I and HNY,6X-G2 linkages which are the major disaccharides found in heparan sulfate (Ernst, et al., (1995), supra, and Linhardt, R., et al., Biochemistry, 29, 2611-2617 (1990)). Heparinase II is capable of recognizing and cleaving both sets of substrate linkages (Ernst, et al., (1995), supra). We have recently identified several residues which are critical to the activity of heparinase I and heparinase II. Cysteine 135 and histidine 203, as well as lysines 198, 199, and 132 of heparinase I were found to be critical to the enzymatic activity of the molecule. Cysteine 348 and histidines 238, 451, and 579 were determined to be crucial for heparinase II activity. (Pending U.S. patent application Ser. No. 09/384,959; Sasisekharan, R., et al., Biochemistry, 34, 14441-14448 (1995); Godavarti, R., et al., Biochemistry, 35, 6846-6852 (1996); Godavarti, R., and Sasisekharan, R., J. Biol. Chem. 273, 248-255 (1998); Shriver, Z., et al., J. Biol. Chem., 273, 22904-22912 (1998); and Shriver, Z., J. Biol. Chem., 273, 10160-10167 (1998)).
Heparinase III is unique in that it is the only member of the heparinase family that recognizes and preferentially cleaves heparan sulfate. Heparinase III also contains no cysteines in its amino acid sequence.
Tumor metastasis involves the spread of tumor cells primarily via the vasculature to remote sites in the body. It is believed that as the extracellular matrix is degraded, the tumor cell-extracellular matrix interactions are disassembled, freeing the tumor cell to extravagate through the capillary bed. Extraordinary progress has been made to elucidate the roles of collagen and related proteins, enzymes (collagenases and others) that degrade the extracellular matrix proteins to regulate tumor angiogenesis and/or tumor cell invasion. It has also recently been hypothesized that HLGAG degrading enzymes, heparinases, assist in the breakdown of the extracellular matrix to regulate tumor growth, angiogenesis and metastasis. It has been suggested that the expression of heparinases in association with tumor development, represents a switch from a metastatic tumor to a non-metastatic tumor and plays a role in initiating the process of metastasis. The hypothesis was reaffirmed by recent cloning of a human heparinase gene and by the demonstration of enhanced malignancy of cancer cells by over-expression of the gene product for heparinase. (Hulett, et al., 1999 and Vlodavsky, et al., 1999).